Two-phase fermentation process for the production of an organic compound

ABSTRACT

The invention relates to a two phase fermentation process for producing an organic compound, in particular an isoprenoid and to a bioreactor comprising a two phase fermentation system for producing an organic compound.

The invention relates to a two phase fermentation process for producingan organic compound, in particular an isoprenoid and to a bioreactorcomprising a two phase fermentation system for producing an organiccompound.

Isoprenoids (also known as terpenoids or terpenes) are a large anddiverse class of naturally occurring organic compounds that findpotential utility, inter alia in the production of pharmaceuticals,cosmetics, perfumes, flavors, animal feed supplements andnutraceuticals.

Conventional production methods involve, e.g., extraction of thesecompounds from plants, microbes and animals. However, these extractionmethods suffer from numerous limitations such as low yield ofextraction, lack of amenability of the source organisms to large scalecultivation and complicated production methods. Also, chemical synthesismethods for producing isoprenoids are not lucrative due to the high costof starting materials and the requirement of extensive productpurification steps. In-vitro enzymatic approaches have also beenexplored but the exploitation of this approach is for instancerestricted by the limited availability of the precursors.

Metabolic engineering of microorganisms for isoprenoid production isbelieved to be most promising for the production of large amounts ofisoprenoids from cheap carbon sources in fermentation processes,although some hurdles still have to be taken (reviewed in Ajikumar etal, Mol. Pharmaceutics, 2008, 5 (2), 167-190). Production of isoprenoidthrough fermentation of microorganisms is deemed more desirable than thetraditional methods as it meets the requirement of sustainableproduction in a more economical, industrially scalable and productiveway. Production of isoprenoids via fermentation methods represents analternate process technology that utilized lower cost of feedstock andhigher productivity, affording a potential for lower cost ofmanufacture.

One of the most frequently encountered problems in fermentationprocedures is end-product inhibition, that is, the microorganismsresponsible for the fermentation may be impaired by the fermentationproduct, e.g. because the fermentation product is cytotoxic.Accumulation of the product beyond a critical concentration inactivatesthe microorganism and substantially diminishes the rate of productivity.This phenomenon is particularly relevant in the production ofisoprenoids as most microbes are destroyed or inactivated in thepresence of these cytotoxic compounds posing a severe limitation toobtain highly productive strains. Owing to these compounds not beingobtained beyond a critical concentration, additional steps ofconcentration and purification may be required rendering the processcumbersome and expensive.

Another major drawback is that isoprenoids, being highly volatileorganic compounds, are poorly soluble in aqueous solutions. Thus, lossof product during fermentation through the off-gas is a major problem inthe development of an economically feasible process (Asadollahi et al,Biotech Bioengin 2007, 99(3): 666-677). In previous studies, terpenoidssynthesized in E. coli were partly lost by evaporation due to theirhighly volatile character (Newman et al., Biotechnol. Bioeng. (2006) 95:684-691).

To overcome the above mentioned drawbacks, attempts have been made toremove the product from the fermentation medium as the fermentationadvances such that the product concentration does not rise to a pointwhere product biosynthesis is inhibited, thus ensuring a sustainedperiod of high rate of productivity. One such attempt utilizes a liquidthat is immiscible with the aqueous fermentation medium but is anextractant for the desired product. The target product partitionsbetween the extractant and the aqueous fermentation medium when the twoare brought into contact, thereby reducing the concentration of theproduct in the aqueous medium. In-situ separation of the releasedproduct being performed in a two-phase fermentation using an organicsolvent as the secondary phase has been attempted to mitigate thedrawbacks of conventional fermentation methods. (Malinowski, BiotechAdvances 2001, 19: 525-538).

In practice, two-phase extractive fermentation systems are complex owingto the unpredictable nature of these systems, particularly for largescale production. One of the most significant challenges is theselection of the right solvent system for a given product/microorganismcombination. A frequently encountered difficulty, for instance is thatmost common water-immiscible solvents are toxic to the microorganismsand/or hazardous rendering them unsuitable for commercial scaleproduction of products such as isoprenoids via fermentation. It has alsobeen encountered that certain solvents form stable emulsions with theaqueous fermentation medium causing difficulties of separation,equipment blockage etc. The selection of a biocompatible organic carriersolvent with favorable partition coefficients is thus crucial for theimplementation of an effective bioconversion in an aqueous-organicbiphasic system (Cruz et al.2004; León et al. 1998). Preferably, thesolvent has other desirable carrier solvent characteristics, such as lowemulsion-forming tendency, chemical, thermal, and biological stability.

Typically, addition of solvents during the fermentation process involvesaddition of large volumes of an organic solvent within a short timeinterval which, e.g., involves the risk of introducing potentialcontaminants into the medium. A loss of sterility of fermentation hasserious consequences impacting production costs, schedules and affectingthe product quality and quantity. Apart from the risk of introducingcontamination, extractants seriously affect the stability of the signalsfrom crucial probes, like e.g., pH-electrode and dissolved oxygen probe,interfering with accurate measurement of important parameters such pHand dissolved oxygen content of the medium. This is more common duringthe employment of organic solvents as extractants since oxygen usuallyhas a higher solubility in organic solvents. Therefore, addition oforganic solvents during fermentation has a serious impact on theaccurate measurement of crucial parameters such as pH and dissolvedoxygen that are required to control the fermentation.

These limitations have hindered the possibilities of usingwater-immiscible solvents during fermentation for industrial scaleproduction of the target products. There is thus a need for anindustrially scalable fermentation process for the production of organiccompounds, in particular isoprenoids in the presence of awater-immiscible solvent. Further, there is a need to produce thesecompounds with a good yield and productivity with a low tendency tobuild up toxic levels of metabolic intermediates.

It is therefore an objective of the present invention to provide afermentation method for producing organic compounds, in particularisoprenoids, that fulfills this need. It is one objective of the presentinvention to specifically address the challenges associated withutilization of water-immiscible solvents in industrial scalefermentation process.

It is a particular objective of the present invention to provide anindustrially scalable and robust method for the production of organiccompounds which permits the utilization of organic solvents as anextractant.

It is a further objective of the present invention to provide anefficient fermentation process for the industrial scale production of anorganic compound, preferably an isoprenoid, employing a liquid-liquidtwo phase system for sustained period of high productivity.

It has now been found that this objective can be realized by adding awater-immiscible organic solvent to an aqueous medium for culturingcells to form a two phase system. An optimized ratio of thewater-immiscible organic solvent to aqueous medium facilitates theselective extraction of the target compound into specific organicextractants during fermentation.

As described, the industrial scale fermentation process is in particularsuited to be used for the production of an isoprenoid, as it aims atsolving the problems generally associated with these highly volatile andcytotoxic compounds. However, the process is equally well suited to beused for the production of other organic compounds that possess similarprocess challenges.

Accordingly, the present invention provides for a two-phase fermentationprocess for the production of an organic compound comprising the stepsof:

a) adding a water-immiscible organic solvent to an aqueous medium forculturing cells to form a two phase system, the ratio ofwater-immiscible organic solvent to aqueous medium being optimized andthe total volume of solvent and medium is at least 10 L;

b) providing the two phase system with an oxygen probe; then

c) performing a calibration of said oxygen probe; then

d) optimizing oxygen tension in said two phase system; then

e) inoculating said two phase system with a microorganism capable ofproducing said organic compound in said oxygen optimized two phasesystem; then

f) measuring and optimizing oxygen tension; and

g) allowing said microorganism to produce said organic compound.

Herein, the term “water-immiscible” refers to the nature of an organicsolvent or organic solvent mixture being incapable or substantiallyincapable of mixing with the aqueous fermentation medium. For thepurpose of this invention, water-immiscible refers to solvents where asignificant proportion of the solvent does not form a solution in water.A suitable water-immiscible solvent is characterized by a high logPvalue (Schewe et al, Appl Microbiol Biotechnol (2009) 83:849-857). Thewater immiscible solvent preferably has a logP value >3, preferably alogP value >4, more preferably a logP value >4.5, most preferably a logPvalue >5.

For the purpose of the invention, the term “biphasic fermentationmedium” or “two phase fermentation medium” is meant to include atwo-phase medium comprising a fermentation medium with the aqueousmedium forming an aqueous phase and a suitable amount of awater-immiscible organic solvent forming an organic phase.

In one particular embodiment, the calibration of the dissolved oxygenelectrode referred to under point c) is carried out as follows: in afirst step, a zero current measurement is performed by using zeroing gelor nitrogen (N2) or carbon dioxide (CO2) calibration gases,alternatively in a sample medium saturated with one of these gases. Theprobe is then mounted into the fermenter and autoclaved, dodecane isadded and the 100% value is determined (second calibration step) aftersaturating with air. In this particular embodiment, the oxygen probe isthus provided to the fermenter before addition of the solvent. Afteraddition of the solvent and saturation of the two-phase system with air,the calibration to 100% dissolved oxygen is performed.

In another particular embodiment, a two point calibration is performedin the two-phase system. A first calibration step is performed in thetwo-phase system which is depleted of oxygen (calibration to 0%dissolved oxygen). After saturation of the two-phase system with air,the second calibration step to 100% dissolved oxygen is performed.

The term “aqueous phase” typically relates to the phase of a biphasicsystem comprising the aqueous fermentation medium which is formed by itscontact with the organic phase. A system is considered aqueous if wateris the only solvent or the predominant solvent (>50 wt. %,preferably >80 wt. %, more preferably >90 wt. %, based on totalliquids), wherein e.g. a minor amount of alcohol or another solvent (<50wt. %, preferably <20 wt. %, more preferably <10 wt. %, based on totalliquids) may be dissolved (e.g. as a carbon source, in case of a fullfermentative approach) in such a concentration that micro-organismswhich are present remain active.

The term “organic phase”, typically relates to the phase of a biphasicmixture comprising the water-immiscible organic solvent which is formedupon its contact with an aqueous fermentation medium. Thewater-immiscible organic solvent may be any solvent. Preferredwater-immiscible organic solvents are selected from the group ofdodecane, lauric acid, oleic acid, n-decane, butyl stearate, olive oil,corn-oil, diisononyl phthalate (DINP), or any combination thereof. Thesesolvents are particularly preferred as they are substantially non-toxicto most industrially employed microorganisms under the processconditions, tend not to form stable emulsions, have good partitioncoefficients for common fermentation products, and can be separated fromthese compounds relatively inexpensively. Hence they possess all thefeatures imperative to render the fermentation viable on an industrialscale. In a particular preferred embodiment, the water-immiscibleorganic solvent is dodecane.

In principle, the production of the isoprenoid can be carried out in amanner based on methodology known per se, e.g. as described in the priorart mentioned herein above. The host cell may be used in a fermentativeproduction of the isoprenoid, or it may be used to produce a monoterpenesynthase or sesquiterpene synthase, which can thereafter then be usedfor synthesis of the desired terpenoid.

Advantageously, the isoprenoid is produced in a fermentative process,i.e. in a method comprising cultivating a host cell in a culture mediumunder conditions wherein typically a monoterpene synthase orsesquiterpene synthase is expressed. The actual reaction catalyzed bythe monoterpene synthase or sesquiterpene synthase typically takes placeintracellular.

It should be noted that the term “fermentative” is used herein in abroad sense for processes wherein use is made of a culture of anorganism to synthesize a compound from a suitable feedstock (e.g. acarbohydrate, an amino acid source, a fatty acid source). Thus,fermentative processes as meant herein are not limited to anaerobicconditions, and extended to processes under aerobic conditions. Suitablefeedstocks are generally known for host cells. Suitable conditions canbe easily found using routine experimentation, using general knowledge,the present patent application and, optionally, other known methodologyas, e.g., for Rhodobacter host cells described in WO 2011/074954 (inparticular page 68, examples, general part, shake-flask procedure) whichis incorporated herein by reference.

In principle, the pH of the reaction medium (culture medium) used in amethod according to the invention may be chosen within wide limits, aslong as it is compatible with the host cell and the isoprenoid synthase(in the host cell) is active and displays a wanted specificity under thepH conditions. The pH is preferably selected such that the cells arecapable of performing their intended function or functions. The pH mayin particular be chosen within the range of four pH units below neutralpH and two pH units above neutral pH, i.e. between pH 3 and pH 9 in caseof an essentially aqueous system at 25° C. Good results have e.g. beenachieved in an aqueous reaction medium having a pH in the range of 6.8to 7.5.

In particular in case a yeast and/or a fungus is used, acidic conditionsmay be preferred, in particular the pH may be in the range of pH 3 to pH8, based on an essentially aqueous system at 25° C. If desired, the pHmay be adjusted using an acid and/or a base or buffered with a suitablecombination of an acid and a base.

Microorganisms often need high levels of oxygen for effective aerobicgrowth. On the other hand, exposure to high oxygen levels can poseoxidative stress upon microorganisms, leading to low productivity andgrowth. Therefore it is often necessary to carefully control the levelof dissolved oxygen in the fermentation broth. For Rhodobacterspaeroides, for instance, a strain that can be used for the productionof isoprenoids, it is advantageous to use high levels of oxygensaturation in an early phase of a fermentation process to generatebiomass and low oxygen saturation in later stages to promote productionof the isoprenoid. Therefore oxygen tension in the broth needs to bemaintained at distinct levels at all times, typically between 10% and100% saturation, preferably 20%-60% in the early phase of fermentationand 0% and 50%, preferably 0% to 25% in the later phase of thefermentation.

In a preferred embodiment, therefore, the oxygen tension (dissolvedoxygen (DO)), is between 50-100%, preferably between 80-100%, morepreferably about 100% at the time of inoculation. Preferably the oxygentension is allowed to decrease to between 10-60%, preferably between20-50%, more preferably between 30-40%, most preferably about 35%shortly after inoculation, and kept at this level during production ofbiomass. During production of the organic compound of interest, thedissolved oxygen tension is preferably kept between 0-50%, morepreferably between 5-25%, more preferably between 10-15%, mostpreferably about 12.5%.

In a working example, the inventors have shown that these conditions areachieved when performing a process according to the invention. Acomparable example wherein dodecane was added after inoculation showsthat these conditions are not achieved and that addition of dodecaneafter inoculation has a negative effect on the fermentation process.

Anaerobic conditions are herein defined as conditions without any oxygenor in which substantially no oxygen is consumed by the cultured cells,in particular a micro-organism, and usually corresponds to an oxygenconsumption of less than 5 mmol/l·h, preferably to an oxygen consumptionof less than 2.5 mmol/l·h, or more preferably less than 1 mmol/l·h.Aerobic conditions are conditions in which a sufficient level of oxygenfor unrestricted growth is dissolved in the medium, able to support arate of oxygen consumption of at least 10 mmol/l·h, more preferably morethan 20 mmol/l·h, even more preferably more than 50 mmol/l·h, and mostpreferably more than 100 mmol/l·h.

Oxygen-limited conditions are defined as conditions in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The lower limit for oxygen-limited conditions is determined bythe upper limit for anaerobic conditions, i.e. usually at least 1mmol/l·h, and in particular at least 2.5 mmol/l·h, or at least 5mmol/l·h. The upper limit for oxygen-limited conditions is determined bythe lower limit for aerobic conditions, i.e. less than 100 mmol/l·h,less than 50 mmol/l·h, less than 20 mmol/l·h, or less than to 10mmol/l·h.

Whether conditions are aerobic, anaerobic or oxygen-limited is dependenton the conditions under which the method is carried out, in particularby the amount and composition of ingoing gas flow, the actualmixing/mass transfer properties of the equipment used, the type ofmicro-organism used and the micro-organism density.

In principle, the temperature used is not critical, as long as theisoprenoid synthase (in the cells), shows substantial activity.Generally, the temperature is at least 0° C., in particular at least 15°C., more in particular at least 20° C. A desired maximum temperaturedepends upon the isoprenoid synthase and the host cell used. Dependingon the cells and/or the isoprenoid synthase used, the temperature is 70°or less, preferably 50° C. or less, more preferably 40° C. or less, inparticular 37° C. or less. Organisms like Thermus thermophilus have atemperature optimum for growth between 49° C. and 72° C., Escherichiacoli of about 37° C., and many fungal microorganisms like yeasts andbacterial microorganisms like Rhodobacter spaeroides have temperatureoptima around 30° C. In case of a fermentative process, the incubationconditions can be chosen within wide limits as long as the cells showsufficient activity and/or growth. This includes pH ranges, temperatureranges and aerobic, oxygen-limited and/or anaerobic conditions.

In one embodiment of the invention, the water-immiscible organic solventis introduced in the aqueous medium in an amount effective to facilitatethe in-situ extraction of the produced organic compound into the organicphase and to increase the rate and/or yield of its production by themicro-organism in the aqueous phase. According to a preferred embodimentof the invention, the ratio of water-immiscible organic solvent toaqueous medium is between 0.5% (v/v) and 60%(v/v), preferably between 2%(v/v) and 40%(v/v) and more preferably between 5%(v/v) and 20% (v/v).

A solvent to be utilized as an extractant in a process according to theinvention preferably meet the following requirements for use in acommercial two-phase extractive fermentation process: low solubility inwater, non-toxic to the producing microorganism, large partitionco-efficient for the product, low partition-coefficient for nutrients,high selectivity, low emulsion forming tendency, high chemical andthermal stability, non-biodegradability, non-hazardous and/or low cost.

In particular suitable (for extraction from an aqueous reaction medium)is extraction with a liquid organic solvent, such as a liquidhydrocarbon. From initial results it is apparent that this method isalso suitable to extract the isoprenoid (or further product) from areaction medium comprising cells according to the invention used for itsproduction, without needing to lyse the cells for recovery of theisoprenoid (or further product),In particular, the organic solvent maybe selected from liquid alkanes, liquid long-chain alcohols (alcoholshaving at least 12 carbon atoms), and liquid esters of long-chain fattyacids (acids having at least 12 carbon atoms). Suitable liquid alkanesin particular include C6-C16 alkanes, such as hexane, octane, decane,dodecane, isododecane and hexadecane. Suitable long-chain aliphaticalcohol in particular include C12-C18 aliphatic alcohols, like oleylalcohol and palmitoleyl alcohol. Suitable esters of long-chain fattyacids in particular include esters of C1-C4 alcohols of C12-C18 fattyacids, like isopropyl myristate, and ethyl oleate

In an advantageous embodiment, isoprenoid (or a further product) isproduced in a reactor comprising a first liquid phase (the reactionphase), said first liquid phase containing cells according to theinvention, wherein the isoprenoid (or a further product) is produced,and a second liquid phase (organic phase that remains essentiallyphase-separated with the first phase when contacted), said second liquidphase being the extracting phase, for which the formed product has ahigher affinity. This method is advantageous in that it allows in situproduct recovery. Also, it contributes to preventing or at leastreducing potential toxic effects of isoprenoid (or a further product) tothe cells, because due to the presence of the second phase, theisoprenoid (or a further product) concentration in the reaction phasemay be kept relatively low throughout the process. Finally, theextracting phase contributes to extracting the isoprenoid (or furtherproduct) out of the reaction phase.

In a preferred method of the invention the extracting phase forms alayer on top of the reaction phase or is mixed with the reaction phaseto form a dispersion of the reaction phase in the extracting phase or adispersion of the extracting phase in the reaction phase. Thus, theextracting phase not only extracts product from the reaction phase, butalso helps to reduce or completely avoid losses of the formed productfrom the reactor through the off-gas, that may occur if isoprenoid isproduced in the (aqueous) reaction phase or excreted into the (aqueous)reaction phase. Isoprenoid is poorly soluble in water and thereforeeasily volatilizes from water. It is contemplated that isoprenoidsolvated in the organic phase (as a layer or dispersion) is at leastsubstantially prevented from volatilization.

Suitable liquids for use as extracting phase combine a lower densitythan the reaction phase with a good biocompatibility (no interferencewith the viability of living cells), low volatility, and near absoluteimmiscibility with the aqueous reaction phase. Examples of suitableliquids for this application are liquid alkanes like decane, dodecane,isododecane, tetradecane, and hexadecane or long-chain aliphaticalcohols like oleyl alcohol, and palmitoleyl alcohol, or esters oflong-chain fatty acids like isopropyl myristate, and ethyl oleate (seee.g. Asadollahi et al. (Biotechnol. Bioeng. (2008) 99: 666-677), Newmanet al. (Biotechnol. Bioeng. (2006) 95: 684-691) and WO 2009/042070). Ina preferred embodiment, a process according to the invention isprovided, wherein the water-immiscible organic solvent is a liquidalkane or a long-chain aliphatic alcohol or an ester of a long-chainfatty acid or an isoprenoid. In a preferred embodiment, the organicsolvent is dodecane, lauric acid, oleic acid, n-decane, butyl stearate,olive oil, corn oil or DINP, most preferably dodecane.

In a process of the invention, the water-immiscible organic solvent isadded prior to the start of the fermentation, i.e. before inoculatingthe two phase system with a microorganism capable of producing theorganic compound. It is an advantage to add the solvent before theinitiation of fermentation since introducing the solvent duringfermentation carries the risk of introducing contaminants into themedium which could be detrimental to the outcome of the fermentationprocess. Additionally, oxygen has a higher solubility in organicsolvents which complicates the accuracy of the measurement if thesolvents are added afterwards. Therefore, addition of organic solventsduring fermentation bears a crucial impact on the accurate measurementof operational parameters such as pH and dissolved oxygen required tocontrol the fermentation. One additional advantage is that the point intime of introducing the solvent in a conventional method is dictated bythe fermentation process.

If desired, isoprenoids produced in a method according to the invention,or a further compound into which isoprenoid has been converted after itspreparation (such as nootkatone), is recovered from the reaction medium,wherein it has been made. A suitable method is liquid-liquid extractionwith an extracting liquid that is non-miscible with the reaction medium.

A particular strategy of conducting the fermentation is by adding theorganic solvent prior to the start of the fermentation, enabling in-situsterilization of the medium and solvent in the absence of the producingmicroorganism. The concurrent sterilization of the two phases furtherevades the need for multiple sterilization steps of the individualphases. In addition, the calibration of the crucial probes such as theoxygen probe must be undertaken in the presence of the organic solvent.Therefore, the oxygen probe must be suitable for measuring oxygentension in the two phase system, thus in the presence of the organicsolvent. By adding the solvent beforehand and optimizing the oxygentension in the two-phase system, the disadvantages associated with theinterim addition of the solvent during fermentation are circumvented. Ina preferred embodiment, the invention provides a process according tothe invention, wherein the process further comprises sterilizing the twophase system prepared in step a), preferably by sterilizing the twophase system at at least 1 bar overpressure for at least 20 minutes atat least 120° C., preferably between 20-40 minutes at about 121° C. atat least 1 bar overpressure. Preferably the sterilization step takesplace before step e), more preferably before step c). In one particularembodiment, instead of sterilizing the two phase system, sterile mediumis used as the aqueous phase, typically sterilized before or afteradding the medium to the bioreactor, and sterile solvent is addedthereto. It is possible to add sterile solvent for instance by passingthe solvent through a sterilization filter (e.g. a 0.22 μm filter) or byusing pre-sterilized solvent. Methods for adding a solvent to an aqueousphase, such that the resulting two-phase system remains sterile areknown in the art.

For the purpose of the invention, the terms “fermentation medium” and“medium” are meant to include the liquid medium in which themicroorganisms are grown. In a particular embodiment it is preferred touse a semisolid medium. A typical fermentation medium commonly includesa substrate and nutrients. The fermentation medium additionally containsthe microorganism, the product produced by the microorganism, metabolicintermediates and other components such as salts, vitamins, amino acids,co-factors and antibiotics. Substrates are commonly sugars or morecomplex carbohydrates that are metabolized by the microorganism toobtain energy and basic structural components, but can also be lipidsand/or proteins.

The two-phase fermentation process of the present invention isparticularly suitable for the industrial scale production of an organiccompound, preferably an isoprenoid. Accordingly, in a preferredembodiment of the invention, the total volume of the solvent and mediumis at least 30 L, preferably at least 100L, more preferably at least1,000 L, more preferably at least 10,000 L, most preferably at least30,000 L or more.

In a preferred embodiment, a process according to the invention isprovided, wherein the organic compound is an isoprenoid. Herein, theterm “isoprenoid” refers to a large and diverse class of naturallyoccurring organic compounds typically composed of two or more units ofhydrocarbons, with each unit consisting of five carbon atoms arranged ina specific pattern. Isoprenoids are built from isoprene units(2-methyl-1,3-butadiene) and the biological precursor for all naturalisoprenoids is isopentenyl diphosphate (IPP). Isoprene (2-methyl-1,3butadiene is a branched-chain unsaturated hydrocarbon. Non-limitingexamples of suitable isoprenoids include hemiterpenes (derived from asingle isoprene unit) such as isoprene, monoterpenes (derived from twoisoprene units) such as mycrene, sesquiterpenes (derived from threeisoprene units) such as amorpha-4,11-diene, diterpenes (derived fromfour isoprene units) such as taxadiene, triterpenes (derived from sixisoprene units) such as squalene, tetraterpenes (derived from eightisoprene units) such as 3-carotene and polyterpenes (derived from morethan eight isoprene units) such as polyisoprene or a mixtures of these.Terpenoids are also included as terpenes for the purposes of the presentinvention. In a particular preferred embodiment, the isoprenoid is amonoterpene or a sesquiterpene, diterpene or triterpene, more preferablya monoterpene or iridoid selected from the group consisting ofAscaridole, Bornane, Borneol, Camphene, Camphor, Cantharidin, Carene,Carvacrol, Carveol, Carvone, Carvonic acid, Chrysanthemic acid,Chrysanthenone, Citral, Citronellal, Citronellol, Cuminaldehyde,P-Cymene, Cymenes, Epomediol, Eucalyptol, Fenchol, Fenchone, Geranicacid, Geraniol, Geranyl acetate, Grapefruit mercaptan, Halomon,Hinokitiol, (S)-Ipsdienol, Levoverbenone, Limonene, Linalool, Linalylacetate, Lineatin, P-Menthane-3,8-diol, Menthofuran, Menthol, Menthone,Menthoxypropanediol, Menthyl acetate, 2-Methylisoborneol, Myrcene,Myrcenol, Nerol, Ocimene, Perilla ketone, Perillaldehyde,Perillartine,Phellandrene, Picrocrocin, Pinene, Alpha-Pinene, Beta-Pinene,Piperitone, Pulegone, Rhodinol, Rose oxide, Sabinene, Safranal,Sobrerol, Terpinen-4-ol, Terpinene, Terpineol, Thujaplicin, Thujene,Thujone, Thymol, Thymoquinone, Umbellulone, and Verbenone, and/or

a sesquiterpene or a sesquiterpene lactone selected from the groupconsisting of Abscisic acid, Amorpha-4,11-diene, Andrographolide,Aristolochene, Artemether, Artemotil, Artesunate, Bisabolene, Bisabolol,Botrydial, Cadalene, Cadinene, Alpha-Cadinol, Delta-Cadinol, Capnellene,Capsidiol, Carotol, Caryophyllene, Cedrene, Cedrol, Copaene, Cubebol,Elemene, Farnesene, Farnesol, Furanolactone, Germacrene, Guaiazulene,Guaiene, Guaiol, Gyrinal, Hernandulcin, Humulene, Illudin, Indometacinfarnesil, Isocomene, Juvabione, Longifolene, Mutisianthol, Nerolidol,Nootkatone, Norpatchoulenol, Onchidal, Patchoulol, Periplanone B,Petasin, Phaseic acid, Polygodial, A-Santalol, B-Santalol, Santonicacid, Selinene, Sterpuric acid, Thujopsene, Valencene, Velleral,Verrucarin A, Vetivazulene, A-Vetivone, and Zingiberene, and/or

a diterpene, a pleuromutilin or a taxane selected from the groupconsisting of Abietane, Abietic acid, Agelasine, Aphidicolin,Beta-Araneosene, Bipinnatin J, Cafestol, Carnosic acid, Cembrene A,10-Deacetylbaccatin, Ferruginol, Fichtelite, Forskolin, Galanolactone,Geranylgeraniol, Gibberellin, Ginkgolide, Grayanotoxin, GuanacastepeneA, Ingenol mebutate, Isocupressic acid, Isopimaric acid, Kahweol,Labdane, Lagochilin, Leelamine, Levopimaric acid, Menatetrenone,Momilactone B, 18-Norabietane, Panicudine, Phorbol, Phorbol12,13-dibutyrate, Phytane, Phytanic acid, Phytol, Pimaric acid,Pristane, Pristanic acid, Prostratin, Pseudopterosin A, Quassin,Retinol, Sclarene, Sclareol, Simonellite, Stemarene, Stemodene, Steviol,Steviol glycoside, Taxodone, 12-O-Tetradecanoylphorbol-13-acetate,Tetrahydrocannabinol-C4, Tetrahydrocannabinolic acid, Totarol, andTriptolide, and/or

a triterpene selected from the group consisting of Absinthin,Acetoxolone, Aescin, Ambrein, Amyrin, Balsaminapentaol, Balsaminol A,Balsaminol B, Betulin, Betulinic acid, Bevirimat, Boswellic acid,Bryoamaride, Carbenoxolone, Celastrol, Corosolic acid, CucurbalsaminolA, Cucurbalsaminol B, Cucurbitane, Cycloartenol, Cycloastragenol,Dammarane, Endecaphyllacin, Ganoderic acid, Ginsenoside, Glycyrrhetinicacid, Glycyrrhizin, Hederagenin, Hemslecin, Hopane, Hopanoids,Karavilagenin E, Lanostane, Lanosterol, Lepidolide, Lupeol,Malabaricane, Maslinic acid, Momordicilin, Momordicin I, Momordicin-28,Momordicinin, Moronic acid, Neokuguaglucoside, Oleanane, Oleanolic acid,2,3-Oxidosqualene, Panaxatriol, Perseapicroside, Protopanaxadiol,Protopanaxatriol, Sapogenin, Squalane, Squalene, Tetranortriterpenoid,Triterpenoid saponin, Ursolic acid, and Yamogenin.

According to a preferred embodiment of the invention, saidmicro-organism, when cultivated in said aqueous phase is capable ofproducing said isoprenoid in an amount sufficient to reach a saturatedconcentration thereof in said aqueous phase.

The present fermentation processes are useful with almost any type ofmicroorganism used for fermentation. The microorganism could be anymicroorganism that is capable of producing an organic compound,preferably an isoprenoid. By way of example, the microorganism can be ayeast, bacteria, fungi, or a mixture of any of these. In a particularpreferred embodiment, the microorganism is optimized for production ofsaid organic compound, for instance by genetic modifications.

In a particular preferred embodiment, the microorganism is a bacteriumor a fungal or a plant cell. In a preferred embodiment, themicroorganism is a bacterial cell selected from the group of Gramnegative bacteria, such as Rhodobacter, Agrobacterium, Paracoccus, orEscherichia;

a bacterial cell selected from the group of Gram positive bacteria, suchas Bacillus, Corynebacterium, Brevibacterium;

a fungal cell selected from the group of Aspergillus, Blakeslea,Penicillium, Phaffia (Xanthophyllomyces), Pichia, Saccharamoyces,Yarrowia, and Hansenula;

a transgenic plant or culture comprising transgenic plant cells, whereinthe microorganism is of a transgenic plant selected from Nicotiana spp,Cichorum intybus, lacuca sativa, Mentha spp, Artemisia annua, tuberforming plants, oil crops and trees; or

a transgenic mushroom or culture comprising transgenic mushroom cells,wherein the microorganism is selected from Schizophyllum, Agaricus andPleurotisi. In a most preferred embodiment, the microorganism is aRhodobacter sphaeroides bacterium.

In one embodiment, a process according to the invention is provided,wherein the process further comprises the isolation of the organiccompound from the two phase system. As the organic compound preferablyaccumulates in the water-immiscible organic solvent, the organiccompound is preferably isolated from said organic solvent. Extractionmethods of organic compounds are known by the skilled person andinclude, but are not limited to liquid-liquid extractions, distillation,pervaporation, etc. In a preferred embodiment, the organic compound isextracted by pervaporation. Pervaporation is a membrane technical methodfor the separation of mixtures of liquids by partial vaporizationthrough a non-porous or porous membrane. It is preferred that before theorganic compound is isolated, the microorganism is allowed to producesaid organic compound for a sufficient amount of time, preferably for atleast 2 days and not more than 7 days.

The process may be performed in a batch mode, in a fed-batch mode or ina continuous mode. The terms “batch mode”, “fed-batch mode” and“continuous mode” are known to one skilled in the art. A processaccording to the invention can easily be performed such that it is runin a batch mode, fed-batch mode or continuous mode.

The invention further provides a bioreactor, which is a vessel or adevice for containing medium and for carrying out a fermentationprocess. The bioreactor is capable of carrying out a two phasefermentation process of the invention and preferably is capable ofproviding the optimum growth conditions for the microorganism.

In one embodiment, the invention provides a bioreactor comprising atleast 10 L of a two phase system comprising a water-immiscible organicsolvent and an aqueous medium for culturing cells, wherein the ratio ofwater-immiscible organic solvent to aqueous medium is between 0.5% (v/v)and 60%, preferably between 2% (v/v) and 40% (v/v), more preferablybetween 5% (v/v) and 20% (v/v), most preferably between 10% (v/v) and20% (v/v). The bioreactor may be of any shape, generally tubular,suitably may be a cylindrical shaped vessel. it is desirable that thebioreactor is made of a material that is inert to the two-phase medium,for example stainless steel or a suitably lined metallic vessel, oroptinally an inert plastic material.

The design of the bioreactor could be of batch-type or fed-batch type.The bioreactor of the invention preferably comprises the major portionsthat include a vessel comprising a reaction port and a harvesting port.

In a preferred embodiment, the bioreactor comprises at least 30 L, morepreferably at least 100 L, more preferably at least 1000 L, mostpreferably at least 10000 L of a two phase system comprising awater-immiscible organic solvent and an aqueous medium for culturingcells.

The bioreactor preferably comprises a means for introducing the aqueousmedium into the vessel and means for introducing the water-immiscibleorganic solvent for contact with the aqueous medium to form a two-phasesystem. Such water-immiscible organic solvents are selected from thegroup consisting of dodecane, lauric acid, oleic acid, n-decane, butylstearate, olive oil, corn oil, and DINP, or any combination thereof.

Because the bioreactor according to the invention is intended forproducing an organic compound, preferably an isoprenoid, using a processaccording to the invention, in a preferred embodiment, the bioreactoraccording to the invention, further comprises a microorganism capable ofproducing an organic compound. Preferably said organic compound is anisoprenoid.

In order to measure oxygen tension, pH and temperature in the two-phasesystem present in said bioreactor, in a preferred embodiment, abioreactor according to the invention further comprises one or moreprobes, capable of measuring these parameters in the two phase system.The bioreactor according to the invention preferably comprises at leastone probe capable of measuring oxygen (oxygen probe).

In one particular embodiment, an organic solvent is present in saidbioreactor. Preferably, the organic solvent is a liquid alkane or along-chain aliphatic alcohol or an ester of a long-chain fatty acid oran isoprenoid, preferably selected from the group consisting ofdodecane, lauric acid, oleic acid, n-decane, butyl stearate, olive oil,corn oil and DINP. In a most preferred embodiment, the organic solventis dodecane.

Preferably the microorganism present in said bioreactor is:

a bacterial cell selected from the group of Gram negative bacteria, suchas Rhodobacter, Agrobacterium, Paracoccus, or Escherichia;

a bacterial cell selected from the group of Gram positive bacteria, suchas Bacillus, Corynebacterium, or Brevibacterium;

a fungal cell selected from the group of Aspergillus, Blakeslea,Penicillium, Phaffia (Xanthophyllomyces), Pichia, Saccharamoyces,Yarrowia, and Hansenula;

a transgenic plant or culture comprising transgenic plant cells, whereinthe microorganism is of a transgenic plant selected from Nicotiana spp,Cichorum intybus, lacuca sativa, Mentha spp, Artemisia annua, tuberforming plants, oil crops and trees; or

a transgenic mushroom or culture comprising transgenic mushroom cells,wherein the microorganism is selected from Schizophyllum, Agaricus andPleurotisi. In a most preferred embodiment, the microorganism is aRhodobacter sphaeroides bacterium. In a particular preferred embodiment,the microorganism is optimized for production of said organic compound,for instance by genetic modifications.

The following examples describe specific embodiments of the presentinvention and are not intended to limit the invention in any way.

LEGEND TO THE FIGURES

FIG. 1: Oxygen profile during fermentation of valencene in a 40 Lfermenter adding n-dodecane prior to inoculation. Prior to addition ofthe seed culture, the oxygen electrode was carefully calibrated at 0%and 100% oxygen saturation. After inoculation, the dissolved oxygen (DO)was allowed to decrease to 35% and was kept constant thereafter. At 70hthe DO was set to 12.5% and kept constant until the end of thefermentation.

FIG. 2: Oxygen profile during fermentation of valencene in a 40 Lfermenter adding n-dodecane after inoculation. Prior to addition of theseed culture, the oxygen electrode was carefully calibrated at 0% and100% oxygen saturation. After inoculation, the dissolved oxygen (DO) wasallowed to decrease to 35% and was kept constant thereafter. At 10 hafter inoculation n-dodecane was slowly added, causing a rapid increasein dissolved oxygen.

EXAMPLES Example 1 Seed Medium

TABLE 1 Ingredient g/L Yeast extract 20.8 MgSO₄•7H₂O 10.3 ZnSO₄•7H2O0.086 MnSO₄•H2O 0.029 CaCl₂•2H₂O 1.08 FeSO₄•7H₂0 0.96 KH₂PO₄ 1.44 K₂HPO₄1.44

pH is adjusted to 7.0 with 5N NaOH

Components (except glucose) are dissolved in water, adjusted to pH 7 andautoclaved.

Example 2 Main Fermentation Medium

TABLE 2 Ingredient g/L Yeast extract 25 MgSO₄•7H₂O 1.5 ZnSO₄•7H₂O 0.1MnSO₄•H₂O 0.03 CaCl₂•2H₂O 1.1 FeCl₃•6H₂O 0.15 K₂HPO₄ 1.5 KH₂PO₄ 1.5(NH₄)₂Fe(SO₄)₂•6H₂O 1.2 (NH₄)₂SO₄ 2.4 (NH₄)H₂PO₄ 1 MgCl•6H₂O 1.7

Example 3 Two Point Calibration of the Oxygen Electrode

The fermentation medium is prepared according to recipe (Table 2) andautoclaved inside the fermenter. Sterile glucose solution is added to afinal concentration of 30 g/L and neomycin (100 mg/ml) is added. Then 20vol % of sterile n-dodecane is added. The stirrer is agitated at themaximum speed applied during fermentation run and the airflow is set toleast 1 vvm (volume/volume per minute). Overpressure is kept as low aspossible and constant over the fermentation run. pH and temperature areset to fermentation values. Nitrogen gas is sparged into the fermenterat 1 vvm until the reactor is fully depleted of oxygen. When the valueread in the electrode is stable, that electrode value is fixed as 0%.

Alternatively, for large fermenters with volume >100 l, calibration to0% oxygen is performed outside of the main fermenter in a small vesseland the calibrated electrode is then transferred to the main fermenter.

The main fermenter is then sparged with air at the maximal gas-flowapplied during fermentation run. When the value read in the electrode isstable, that electrode value is fixed as 100%. Other fermentationparameters (pressure, air flow, rpm) are set to fermentation start pointand the fermenter is inoculated with a Rhodobacter sphaeroidespreculture with a cell density corresponding to ca. 40 OD620 nm unitswith an inoculation ratio of 5%.

Example 4 Comparative Example Adding n-dodecane during Fermentation

The main fermentation was performed in a 35 m³ vessel that is chargedwith 10000 kg medium containing 22 g/L initial glucose at a temperatureof 30° C., a pH of 7.0 (controlled with 28 wt % NH₃ solution), anaeration of 2 Nm³/min and an overpressure of 0.5 bar. At 0 hours themain fermentation was inoculated using 1 m³ seed culture (see example7). The dissolved oxygen (DO) is kept constant at 35% by adjusting thestirrer speed between 60 and 90 RPM and adjusting the aeration between0.2 and 1.8 vvm. After 12 hours of batch fermentation the pO₂ valuedecreased strongly to a value of 15% and started to increase rapidlyafter that. Also the pH increase rapidly from pH 7.0 to pH 7.7. Analysisshows that all glucose was consumed and glucose feeding was started,keeping the pH at 7.0. After 26 hours 2000 L n-dodecane was slowly addedto the fermenter via a sterile filter, with a dosing rate of 500 L/hour.During addition of n-dodecane it was observed that the pO₂ signal wasvery unstable and difficult to control. Finally, after all n-dodecanewas added the pO₂ value had increased from 35% to about 80%. Regularlyresidual glucose checks demonstrated that the process was proceeding inglucose limitation. After about 60 hours the respiration rate started todecrease and glucose accumulation started to occur. After 78 hoursglucose was no longer consumed and the concentration rapidly increased.Analysis of the valencene concentration indicated that the biomass wasno longer active.

Example 5 Valencene Fermentation in a 40 L Fermenter Adding n-dodecanePrior to Inoculation

A frozen stock vial containing 1 mL of Rhodobacter sphaeroides valenceneproduction strain containing genes encoding the mevalonate pathway ofParacoccus denitrificans and a Valencene synthase gene (see Example 9)was cultivated in a 2000 mL shake flask containing 500 mL medium asdescribed in example 1 and an additional 100 mg/L neomycin. The shakeflask was incubated on an orbital shaker at 180 rpm and an amplitude of4 cm for 46 hours at 30° C. The culture was transferred into a 40 Lstainless steel Techfors-S fermentor from Infors that containing 9 kg ofmedium with the following composition: 21 g/l yeast extract, 1.73 g/lMgSO₄.7H₂O, 0.104 g/l ZnSO₄.7H₂O, 0.035 g/l MnSO₄.H₂O, 1.3 g/lCaCl₂.2H₂O, 1.73 g/l KH₂PO₄, 1.73 g/l K₂HPO₄, 33 g/l dextrose, 1.2 g/l(NH₄)₂Fe(SO₄)₂.6H₂O, 0.17 g/l FeCl₃.6H₂O, 2.76 g/l (NH₄)₂SO₄, 0.94 g/l(NH₄)H₂PO₄, 1.6 g/l MgCl6H₂O and 125 g/l n-dodecane. Prior to additionof the seed culture the medium was sterilized, adjusted to pH 7 withNH₄OH 25 wt % and the oxygen electrode was carefully calibrated at 0%and 100% oxygen saturation, according to the procedure described inexample 2. After inoculation the fermentation runs in batch mode at 30°C. and pH at 7.0 for 16.5 hours, allowing the DO to decrease initiallyto 35% and keeping in constant thereafter. After this batch phase theglucose feed is started by adding 17.2 kg of a solution containing 55 wt% glucose. During this phase the pH was kept constant at 7.0 and the DOat 35%. At 70 hours the pO₂ setpoint was put on 12.5% and kept constantuntil the end of fermentation by adjusting the stirrer speed. The oxygenprofile during fermentation was recorded and is presented in FIG. 1.

Example 6 Comparative Example Valencene Fermentation in a 40 L Fermenter

The experiment described in example 5 was repeated except that themedium of the main fermentation did not contain n-dodecane. Afterpreparation, sterilization and cooling of the medium the oxygenelectrode was carefully calibrated at 0% and 100% oxygen saturation. Thefermenter was then inoculated by adding 500 ml of a seed cultureprepared according to the procedure described in example 4 and run inbatch mode at 30° C. and pH 7.0 for about 10 hours, allowing the DO todecrease to 35%. At 10 hours after inoculation the glucose feed wasstarted, followed by the slow addition of 1500 ml n-dodecane via asterile filter, with a dosing rate of 400 ml/hour. After adding then-dodecane the DO rapidly increased as shown in FIG. 2. Analysis of theresidual glucose showed that after about 20 hours of fermentationglucose started to accumulate. Analysis of the amount of biomassindicated that cell growth had stopped.

Example 7 Seed Culture

A 2500 L fermenter is charged with 1000 kg seed medium. The mediumcomposition is shown in table 1. After sterilization at 121° C. for 30minutes, neomycin is added via a sterile filter to a final concentration100 mg/kg and 68 kg 55 wt % glucose. Next, the oxygen probe iscalibrated followed by inoculation of the fermentor with 500 mL of acell culture. The fermentation is operated in batch mode for 48 hours at30° C., pH 7.0 using 25 wt % NH₃ solution, an aeration of 1 vvm, anoverpressure of 0.3-0.4 bar, and an DO of 35%. The DO is kept constantby agitation.

Example 8

Main Fermentation Adding n-dodecane Prior to Inoculation

The main fermentation runs in fed-batch mode using a 35 m³ vessel thatis charged with 10000 kg medium. The medium composition is shown intable 2. To prepare 10000 kg of medium, medium components are dissolvedin water, sterilized by autoclaving at 121° C. for 30 minutes in thebioreactor.

After sterilization and cooling of the medium the pH is adjusted to 7with NH₄OH 25%. After sterilization 600 kg 55 wt % glucose is added.Next 2000 L (1666 kg) n-dodecane is added via a sterile filter. Theoxygen probe is calibrated (see example 3). The fermentation starts byadding 1000 L culture obtained from a seed. After inoculation thefermentation runs in batch mode at 30° C. and pH at 7.0 for 20 hours,keeping the DO at 35%. The OD is kept constant at 35% by adjusting thestirrer speed between 60 and 90 RPM and adjusting the aeration between0.2 and 1.8 vvm. After 20 hours the glucose feed is started, keeping thepH at 7.0 and the DO at 35%. At 70 hours the pO₂ setpoint was put on12,5% and kept constant until the end of fermentation by adjusting thestirrer speed. The fed-batch mode runs for approximately 120 hours at30° C. and pH at 7.0.

Regularly residual glucose checks demonstrated that the process wasproceeding in glucose limitation. The respiration rate remained highover the whole course of the fermentation and no glucose accumulationwas observed. Analysis of the valencene concentration showed that thebiomass was still producing valencene 140 h after inoculation .

Example 9 Valencene Producing Rhodobacter Strain

Rhodobacter sphaeroides strain Rs265-9c was obtained from Rhodobactersphaeroides strain ATCC 35053 [purchased from the American Type CultureCollection (ATCC—Manassas, Va., USA—www.atcc.org); number 35053;Rhodobacter sphaeroides (van Niel) Imhoff et al., isolated from a sewagesettling pond in Indiana and deposited as Rhodopseudomonas sphaeroidesvan Niel] after two rounds of mutagenesis and was used as the base hostfor construction of recombinant strains having improved production ofisoprenoid. For details about this strain, see WO20110749654, which isincorporated herein by reference.

1. A two-phase fermentation process for the production of an organiccompound, preferably an isoprenoid, comprising the steps of: a) adding awater-immiscible organic solvent to an aqueous medium for culturingcells to form a two phase system, wherein the ratio of water-immiscibleorganic solvent to aqueous medium is between 0.5% (v/v) and 60% (v/v)and the total volume of solvent and medium is at least 10 L; b)providing the two phase system with an oxygen probe; then c) performinga calibration of said oxygen probe; then d) optimizing oxygen tension insaid two phase system; then e) inoculating said two phase system with amicroorganism capable of producing said isoprenoid in said oxygenoptimized two phase system; then f) measuring and optimizing oxygentension; and g) allowing said microorganism to produce said organiccompound.
 2. Process according to claim 1, wherein the total volume ofsolvent and medium is at least 30 L, preferably at least 100 L, morepreferably at least 1,000 L, more preferably at least 10,000 L, mostpreferably 30,000 L or more.
 3. Process according to claim 1, whereinthe process further comprises sterilizing the two phase system preparedin step a), preferably by sterilization for at least 20 minutes at atleast 120° C., wherein the sterilization takes place before step e),preferably before step c).
 4. Process according to claim 1, wherein theprocess further comprises the isolation of the organic compound from thetwo phase system, preferably from the water-immiscible organic solvent.5. Process according to claim 1, wherein the water-immiscible organicsolvent is a liquid alkane or a long-chain aliphatic alcohol or an esterof a long-chain fatty acid or an isoprenoid.
 6. Process according toclaim 1, wherein the water-immiscible organic solvent is dodecane,lauric acid, oleic acid, n-decane, butyl stearate, olive oil, corn oilor DINP, preferably dodecane.
 7. Process according to claim 1, whereinthe microorganism is a Gram positive bacterial cell, a Gram negativebacterial cell, a fungal cell, a transgenic plant or culture comprisinga transgenic plant cell or a transgenic mushroom or culture comprising atransgenic mushroom cell.
 8. Process according to claim 1, wherein thepercentage of water-immiscible organic solvent in the two-phase systemis between 2% (v/v) and 40% (v/v), preferably between 5% (v/v) and 20%(v/v), most preferably between 10% (v/v) and 20% (v/v).
 9. Processaccording to claim 1, wherein the organic compound is an isoprenoid,preferably a monoterpene, a sesquiterpene, a diterpene or a triterpene.10. Process according to claim 1, wherein the dissolved oxygen duringthe production phase step (f) never exceeds 80%, preferably 60%, morepreferably 40%, most preferably 20%.
 11. Bioreactor comprising at least10 L of a two phase system comprising a water-immiscible organic solventand an aqueous medium for culturing cells, wherein the ratio ofwater-immiscible organic solvent to aqueous medium is between 0.5% (v/v)and 60%.
 12. Bioreactor according to claim 11, further comprising amicroorganism capable of producing an organic compound, preferably anisoprenoid.
 13. Bioreactor according to claim 11, further comprising anoxygen probe.
 14. Bioreactor according to claim 1, wherein the organicsolvent is a liquid alkane or a long-chain aliphatic alcohol or an esterof a long-chain fatty acid or an isoprenoid, preferably dodecane, lauricacid, oleic acid, n-decane, butyl stearate, olive oil, corn oil or DINP,more preferably dodecane.
 15. Bioreactor according to claim 1, whereinthe microorganism is a Gram positive bacterial cell, a Gram negativebacterial cell, a fungal cell, a transgenic plant or culture comprisinga transgenic plant cell or a transgenic mushroom or culture comprising atransgenic mushroom cell.